PROJECT SUMMARY The long term goals of my laboratory are to generate new insights into the role of the inflammatory response in tissue repair and to develop novel therapies that shape the inflammatory response to improve tissue repair. For the past 15 years, the major focus of my research has been the role of macrophages in tissue damage, repair and regeneration. My laboratory has published findings that macrophages play important roles in repair of both skin and skeletal muscle and that these cells display tremendous phenotypic plasticity while helping to guide a wound through each phase of healing. Our data also demonstrate that macrophage dysregulation contributes to chronic inflammation and impaired skin wound healing in diabetes as well as impaired healing of traumatic muscle injuries. Importantly, we have demonstrated and that targeting macrophages can induce resolution of inflammation and improve healing. For example, we have found that repurposing the anti-diabetic drug glyburide into a topical treatment for skin wound healing can be used to inhibit the NLRP3 inflammasome in wounds, which results in a switch from a destructive pro-inflammatory to a pro-healing macrophage phenotype and improved healing in diabetic mice. We are now in the process of obtaining an Investigational New Drug designation for our new formulation that is needed to move forward into a Phase I human trial on topical glyburide for diabetic leg ulcers. Following this model pipeline from discovery to clinical trial will be a primary goal of all our future studies. Despite these advances, much remains to be learned about the plasticity of macrophages during tissue repair and the factors that regulate macrophage function, including cell-intrinsic and cell-extrinsic pathways. To this end, my laboratory is studying the contributions of each developmental stage of the monocyte/macrophage lineage to the regulation of wound macrophage function during repair of skin and skeletal muscle, in both normal healing and impaired healing models, in mice and humans. A goal of my laboratory over the next 5 years is to take advantage of newly developed single cell analysis techniques to determine, using unbiased methods, the actual phenotypes that macrophages adopt during normal and impaired wound healing. This approach will help overcome a significant barrier to progress in the field, which is the widespread use of biased and oversimplistic methods to categorize macrophage populations in vivo, and has potential to identify novel macrophage populations involved in healing. We will also use the single cell analysis techniques to guide mechanistic experiments to elucidate the transcription factors and pathways involved in the regulation of different macrophage populations. As our research progresses, we plan to address additional significant gaps in understanding in the wound healing field, including the role of wound pathogens in the (dys)regulation of macrophage function and tissue repair. The overall vision guiding our approach is that by identifying novel regulators of macrophage function during wound healing, we can develop new approaches to manipulate inflammation and improve healing of poorly healing wounds, and in the process, generate a pipeline of candidate therapies to translate into clinical studies.